This work has relevance to our understanding of mechanisms and potential therapies for disorders of brain development. PITX2 is a member of the paired-like class of transcription factors that regulate expression of multiple other genes, through direct binding to DNA. In humans, PITX2 is mutated in Rieger syndrome, a multiple congenital anomaly syndrome affecting eyes, umbilicus, and teeth, with variable abnormalities in the heart, pituitary, and brain. PITX2 is produced in embryonic and adult mammalian brain neurons, but its function in these cells is unknown. In mice, loss of PITX2 function results in abnormal development of neurons in the subthalamic nucleus and superior colliculus, but the exact requirements for Pitx2 in neuronal progenitor proliferation and differentiation are not known. Homozygous loss of Pitx2 is lethal in midgestation due to severe defects of thoracic and abdominal organs, limiting the use of these mice for analysis of PITX2 function in the brain at later time points. PITX2 is expressed in mice from two different promoters as three separate isoforms (a, b, c) that exhibit unique functions in cardiac and craniofacial development;the relative contributions of these different PITX2 isoforms to brain development have not been determined. Our prior studies identified PITX2 expression in discrete neuronal populations in the developing mouse brain, and demonstrated disrupted neuronal development in the subthalamic nucleus and superior colliculus with loss of Pitx2 function. Our global working hypothesis is that PITX2 regulates expression of genes required for one or more aspects of neuronal differentiation, including migration and axon outgrowth, in a brain region-specific and isoform-specific manner. Proposed studies will examine the production, migration, axon formation, and cell fate of developing PITX2 mutant neurons, using existing PITX2cre and isoform specific PITX2 loss of function alleles and a novel PITX2-TaulacZ knock-in allele. Results obtained from these experiments will impact our understanding of PITX2- mediated transcriptional mechanisms that regulate neuronal differentiation and survival. Proposed studies will also uncover PITX2 functions in neuronal differentiation and survival in other regions of the mouse brain, thereby providing crucial insights into the potential role(s) for PITX2 in Rieger syndrome and other developmental brain disorders. These results will help guide future experiments aimed at directing neural stem cell differentiation toward specific neuronal cell subtypes.